Viruses are important etiological agents of a wide variety of diseases. In animals the immune response comprises one of the basic mechanisms to fight viral infections. Classically, the immune response encompasses two facets: the B-lymphocyte antibody response, referred to as humoral immunity and a T-lymphocyte-mediated response, known as cell-mediated immunity. The present application is concerned particularly with the antibody response.
While specific antibody of classes IgG, IgM and IgA can bind to any accessible epitope on a surface protein of a virion, only those antibodies which bind with reasonably high avidity to particular epitopes on a particular protein in the outer capsid or envelope are capable of neutralizing the infectivity of the virion. These are termed "neutralizing antibodies." "Neutralization" as used throughout the instant specification is intended to include not only (1) classical virus neutralization which results when antibody binds to a surface antigen of a virion which ordinarily binds to a receptor on the surface of a susceptible cell and thereby prevents infection of a susceptible cell or leads to opsonization but also includes (2) interactions such as the binding of an antibody to neuraminidase of influenza virus (IF) which results in inhibition of release of progeny virus particles from the plasma membrane of infected cells and slows virus spread and (3) binding of an antibody to fusion protein (F) of paramyxoviruses which does not prevent initiation of infection but does block the direct cell to cell spread of newly formed virions once infection has been established.
Antibodies directed against irrelevant or inaccessible epitopes of surface proteins, or against internal proteins of the virion, or virus-coded non-structural proteins, such as virus-encoded enzymes can sometimes exert indirect immunopathological effects, but may play no role in elimination of the infection. These are "non-neutralizing antibodies." In fact, certain non-neutralizing antibodies not only form damaging circulating "immune complexes" but may actually impede access of neutralizing antibody and enhance the infectivity of the virion for some cells. For example, in the presence of sub-neutralizing concentration of neutralizing antibody or excess of non-neutralizing antibodies viruses such as togaviruses are actually taken up more efficiently by macrophages (via Fc receptors on the macrophage to which the virus-antibody complex binds). The virus multiples intracellulary to high titer inside the macrophages. Hence the non-neutralizing antibodies act as "enhancing antibody." Specific examples of such viruses include dengue virus types 1-4.
Thus in response to a viral infection, two very different kinds of antibodies are produced: neutralizing antibodies and non-neutralizing antibodies. Each is present in the serum of infected individuals or individuals previously exposed to a virus or a viral antigen in varying amounts. In order to assess the true immunocompetent status of an individual it is necessary to know the absolute and relative amounts of both neutralizing and non-neutralizing antibodies. Yet conventional serological assays of antiviral antibodies do not, and in fact cannot, distinguish these two kinds of antibodies. Conventional serological assays measure the presence of both types of antibodies. Hence there is no serological method for measuring or assessing the true immune status or immunocompetence of individuals.
The only conventional method for assessing virus neutralizing ability of serum of individuals has been the virus neutralization assay such as that described by Krech et al., Z. Immuno., Forsch. Bd. 141 S: 411-29 (1971). Neutralization assays require: (1) use of infectious virus and (2) cell culture techniques. Such assays are slow, cumbersome, labor intensive and expensive. Hence there has been a long-felt need for a rapid, inexpensive accurate serological method to assess the immunocompetent status of individuals.
Examples of specific situations in which a rapid, easy test for assessing the immunocompetence of an individual is particularly important include, but are not limited to, the following. Firstly, exposure of a pregnant female to a virus such as rubella virus or cytomegolovirus poses significant risk of congenital defects for the fetus. Using conventional serological methods such as ELISA assays the titer of all IgM and IgG antibodies against the relevant virus, both neutralizing and non-neutralizing, may be determined. If the total IgM level is elevated indicating that the response is due to reaction by a presumably "naive" immune system, a therapeutic abortion will be recommended because it is unlikely that neutralizing antibodies against the virus are present. If, however, only the total IgG level is elevated, no therapeutic abortion will be recommended because the test cannot distingush whether the IgG's present are neutralizing or non-neutralizing antibodies. The patient is faced with a long stress-filled pregnancy which may end in a child with congenital defects.
Secondly, exposure of (or reactivation of previous infection associated with immunosuppression) organ transplant or bone marrow transplant patients to viruses such as cytomegalovirus (CMV) poses significant risks of clinical disease states including pneumonia, hepatitis, retinitis, encephalitis, etc. Moreover, the glomerulopathy induced by CMV adversely affects the survival of kidney grafts, so that renal transplant patients face additional life-threatening organ rejection [see generally, White et al., eds., in Medical Virology, 3d ed., Academic Press, Inc., New York, pp. 419-426 (1986)].
Thirdly, viral infections pose significant, indeed often life-threatening risks for immuno-suppressed patients including cancer patients undergoing chemotherapy, and those afflicted with either congenital or acquired immunodeficiency such as acquired immune deficiency syndrome (AIDS).
Fourthly, certain viral infections endemic to specific geographic areas pose significant risks, for example, for military or diplomatic personnel stationed in these areas. Specific examples include but are not limited to Rift Valley fever, dengue etc. Vaccines may protect by actively eliciting the production of neutralizing antibodies. Evaluation of the immunocompetent status of personnel to be sent to these areas following vaccination is important.
In all the above examples, there is a need for rapid, serological methods for determining both the presence and titer of virus neutralizing antibodies. Examples of formats useful in such rapid, serological methods include but are not limited to Enzyme-Linked Immunosorbent Assays (ELISA), radioimmunoassays (RIA), immunofluoresence or other fluorescence-based assays, agglutination assays, etc.
Hagenaars et al., J. Virol. Methods 6: 233-39 (1983) described a modified inhibition ELISA assay which showed some correlation between ELISA titers and neutralization assay titers for polio virus type I. Unlike the presently described assays, however, the modified inhibition ELISA of Hagenaars et al. was more complex and cumbersome.
Dreesman et al., Virol. 69:700-09 (1976) investigated the site associated with hemagglutinating activity of adenovirus following oxidation of viral antigen and purified virus preparations. Animals immunized with oxidized preparations showed significantly decreased haemgglutination -- inhibiting antibody; however, neutralizing antibody titers were not substantially affected. In contrast, according to the present invention, mild oxidation of the oligosaccharide moiety of virus, viral antigen and virus fragments not only enhances the ability of the immunogen to elicit neutralizing antibody production, but more critically also elicits a protective immune response.